A variety of analytical techniques are used to characterize interactions between molecules, particularly in the context of assays directed to the detection of biomolecular interactions. For example, antibody:antigen interactions are of fundamental importance in many fields, including biology, immunology and pharmacology. In this context, many analytical techniques involve binding of a “ligand” (such as an antibody) to a solid support, followed by contacting the ligand with an “analyte” (such as an antigen). Following contact of the ligand and analyte, some characteristic is measured which is indicative of the interaction, such as the ability of the ligand to bind the analyte. After measurement of the interaction, the ligand:analyte pair is typically disrupted with an elution and/or regeneration solution in order to regenerate surface-bound ligand for further analytical measurement.
The freed analyte of the ligand:analyte pair, however, is commonly not reused; rather, the freed analyte is typically disposed of together with the elution and/or regeneration solution. This practice is undesirable because researchers very often have only limited quantities of the analyte for analytical measurement purposes, and because researchers very often desire to perform further analytical measurements directed to the analyte itself. Accordingly, there is a need in the art to effectively consolidate freed analyte from a ligand:analyte pair such that the freed analyte is amenable to subsequent analytical measurement.
The need to effectively consolidate freed analyte for subsequent analytical measurement may be illustrated in the context of biosensors which use surface plasmon resonance (SPR) to monitor the interactions between an analyte and a ligand bound to a solid support. In this regard, a representative class of biosensor instrumentation is sold by Biacore AB (Uppsala, Sweden) under the trade name BIAcore® (hereinafter referred to as “the BIAcore instrument”). The BIAcore instrument includes a light emitting diode, a sensor chip covered with a thin gold film, an integrated microfluidic cartridge and photo detector. Incoming light from the diode is reflected in the gold film and detected by the photo detector. At a certain angle of incidence (“the SPR angle”), a surface plasmon resonance wave is set up in the gold layer, which is detected as an intensity loss or “dip” in the reflected light. The theoretical basis behind the BIAcore instrument has been fully described in the literature (see, eg., Jönsson, U. et al., Biotechniques 11 620-627 (1991)).
In addition to SPR analysis using the BIAcore instrument, researchers are beginning to appreciate the synergistic effects of coupling SPR technology with other analytical techniques. In this context, the real-time interaction analysis offered by the BIAcore instrument complements other known methods for investigating both biomolecular structure and function. For example, SPR has recently been coupled with mass spectroscopy (i.e., SPR-MS) to provide an extremely powerful micropreparative technique for biomolecular investigations (see, e.g., PCT International Publication No. WO 97/09608). In connection with SPR-MS, analyte is freed from the surface-bound ligand by matrix-assisted laser desorption/ionization for subsequent analytical measurement by mass spectrometry.
One of the problems posed by eluting analyte away from surface-bound ligands for subsequent analytical measurements is that substantial amounts of analyte can be lost due to nonspecific binding of analyte to the walls and other components of the microfluidic cartridge as the elution and/or regeneration solution flows through the microfluidic cartridge. Moreover, once eluted away from surface-bound ligands, analyte must still be consolidated so that there will be enough sample for subsequent analysis. Accordingly, there is a need in the art for improved methods and micropreparative techniques for consolidating biomolecules associated with surface-bound ligands. The present invention fulfills these needs, and provides further related advantages.